Zone electrophoresis in capillaries has become an important technique in the repertoire of liquid-phase separations. See Jorgenson et al., Science 242 (1983); Gordon et al., Science 242 (1988), 224-228; Ewing et al., Anal. Chem. 61 (1989), 292A-303A; Wallingford et al., Advances in Chromatography 29 (1989), 1-76; and Kuhr, Anal. Chem. 62 (1990), 403R-414R. Capillary electrophoresis has been used for separations of small and large molecules and comprises several subtechniques including capillary zone electrophoresis (CZE), capillary gel electrophoresis, micellar electrokinetic capillary chromatography, and capillary isoelectric focusing.
A major aspect of CZE in need of new development is detection; specifically, there is a critical need for detectors capable of responding to the small quantity of sample component in the effective detection volume. Detection schemes developed to date include direct and indirect UV absorption (Hjerten, J. Chromatogr. 347 (1985), 191-198 and Hjerten et al., J. Chromatogr. 3 (1987), 47-61), fluorescence (Jorgenson et al., Anal. Chem. 53 (1981), 1298-1302) and Kuhr et al., Anal. Chem. 60 (1988), 2642-2644) and radioisotope (Pentoney et al., Anal. Chem. 61 (1989), 16421647), as well as mass spectrometric (Smith et al., Anal. Chem. 60 (1988), 436-441; Lee et al., Biomed. Environ. Mass Spectrom. 18 (1989), 844-850; Moseley et al., Chromatogr. 480 (1989), 197-210; and Caprioli et al., J. Chromatogr. 480 (1989), 247-258) and electrometric (Mikkers et al., J. Chromatogr. 169 (1979), 11-20; Huang et al., Anal. Chem. 59 (1987), 2747-2749; and Wallingford et al., Anal. Chem. 59 (1987), 1762-1766) detectors.
However, because CZE employs extremely high potential fields (typically 300 V/cm) to achieve highly efficient separations, detection schemes for CZE are designed to prevent the high potentials used from interfering with the detection process. For example, existing electrical and electrochemical detectors for CZE use elaborate on-column and post-column detection schemes to prevent such interference. One scheme involves construction of 40 .mu.m-diameter holes in the capillary using a laser. Thereafter, small platinum wire electrodes are placed in these holes to carry out on-column conductivity detection. It has been demonstrated that the exact placement of these electrodes on opposite sides of the capillary is critical to minimize noise associated with the high potential field used for separation (Huang et al., Anal. Chem. 59 (1987), 2747-2749). In U.S. patent application Ser. No. 443,059, filed Nov. 28, 1989 and now abandoned, by Zare et al. (continued in Ser. No. 744,642, filed on Aug. 8, 1991 and issued as U.S. Pat. No. 5,223,114 on Jun. 29, 1993), on-column conductivity detectors were disclosed wherein on-column sensing electrodes are located contiguous with the exit of the separation microcolumn. The sensing electrodes must be carefully aligned and an isolation transformer must be used in measuring the conductance (Huang et al., Anal. Chem. 59 (1987), 2747-2749; and Everaerts et al., Isotachophoresis, Journal of Chromatography Library 6, Elsevier: Amsterdam, 1976).
Turning to another detection scheme, Huang et al. recently reported the use of an end-column structure for conductimetric and amperometric detection for CZE in which the sensing electrode is placed at the outlet of the fused-silica capillary (Huang et al., Anal. Chem. 63 (1991), 189-192). While such end-column detectors (also described in U.S. Pat. No. 5,126,023, issued on Jun. 30, 1992 to Huang et al.) demonstrate sensitivities that approach those of previous on-column conductivity detectors, the end-column structures require carefully matched microplumbing in which the analytical capillary is placed inside a second capillary that has an inside diameter slightly larger than the outside diameter of the analytical capillary. For conductimetric detection, epoxy is used in such structures to help maintain structural integrity of the electrode. However, if the epoxy becomes exposed to the electrolyte, it may affect measurements.
Therefore, although good results have been obtained with current detection systems, these systems are limited by their need for painstaking alignment and precise manipulation in situating the various sensing electrodes relative to the separation column. Such limitations result in structures that are difficult to fabricate, expensive and, often times, unreliable. These limitations are especially problematic in electrochemical detectors which are easily fouled during normal use and require frequent cleaning or changing of electrodes. After an electrode is changed, the repeatability of results is very questionable. These problems have limited the routine application of both on-column and post-column modes of electrochemical detection in CZE. See Ewing et al., Anal. Chem. 61 (1989), 292A-303A; Kuhr, Anal. Chem. 62 (1990), 403R-414R; Huang et al., Anal. Chem. 61 (1989), 766-770; Huang et al., J. Chromatogr. 425 (1988), 385-390; Huang et al., J. Chromatogr. 480 (1989), 285-288; Wallingford et al., Anal. Chem. 60 (1988), 1972-1975; Wallingford et al., Anal. Chem. 60 (1988), 258-263; Wallingford et al., J. Chromatogr. 441 (1988), 299-309; and Wallingford et al., Anal. Chem. 61 (1989), 98-100. Additionally, the alignment limitation has precluded the use of existing detectors with automated systems in which the capillary column is automatically connected to and disconnected from the detector. There is a further detection scheme that has yet to be employed in CZE, namely, fiber optic detection (i.e., with chemiluminescence). For example, the use of fiber optics for chemical analysis has been described by Abdel-Latif et al., Anal. Chem. 60 (1988), 2671-2674. It would be useful to develop a fiber optic detection system for CZE such that a minimally dimensioned optical fiber could be used to accurately detect analyte concentration at the end of the capillary column.